The neocortex constitutes the larger part of the brain in mammals and is the primary site of mental functions. No unitary theory of how the cortex works exists. Nevertheless, the basic structure of the cortex develops in stereotyped fashion, is similar in different parts of the cortex and in different mammals, and has not changed much in evolution since its appearance. Because of this, it is conceivable that a "canonical" cortical microcircuit may exist and implement a basic algorithm. Anatomical and physiological studies have suggested that the synaptic connectivity of the cortical microcircuitry is complex, but not random. In the previous cycle of the award we developed an optical method using calcium imaging of slices, to track excitatory circuits in neocortical slices. With this "optical probing" method we have reconstructed synaptic circuits in layer 5 from mouse primary visual cortex (V1) and have discovered extraordinary target specificity in several projections from layer 5 pyramidal neurons. These circuits were precise and identical in different animals suggesting that the neocortex is indeed built out of scores of precise circuits with dedicated junctions. For this next cycle we propose a "frontal attack" on the cortical microcircuitry of mouse V1 using a large-scale optical probing effort with the goal to achieve a relatively complete reconstruction of the inter- and intralaminar excitatory circuitry. We will test whether the precision found in layer 5 applies to other neocortical excitatory connections, as well as search for general rules in this circuit diagram. Our second goal is to test whether there are indeed canonical microcircuits, by reconstructing layer 5 circuits in mouse somatosensory cortex (S1) and compare them with those in V1. In these experiments we will use transgenic mice strains that express GFP in subpopulations of neurons, a novel two-photon stimulation method that enables us to stimulate at will any neuron in the field of view, and exploratory microarray studies to find clusters of genes specifically expressed on subtypes of cortical cells. These basic studies will shed light on the structure of the functional units of the cortex and contribute to build bridges between system and cellular, molecular and biophysical level studies of visual cortex. In addition, they will help understand the central pathophysiological consequences of amblyopia and strabismus and improve analysis of visual evoked potentials and early diagnosis of visual pathologies.